Slow Releasing H2S Donor-Thioglycine Exerts Cardioprotective Effects in Myocardial Ischemia/ Reperfusion in Vivo

Ciro Coletta1, Sofia-Iris Bibli2, Zongmin Zhou3, Athanasia Chatzianastasiou4, Athanassios Giannis5, Efstathios K. Iliodromitis6, Ioanna Andreadou2, Csaba Szabo1, Andreas Papapetropoulos3. 1University of Texas Medical Branch, Department of Anesthesiology, Galveston, Texas, USA, 2University of Athens, Department of Pharmacy, Athens, Greece, 3University of Patras, Department of Pharmacy, Laboratory of Molecular Pharmacology, Patras, Greece, 4University of Athens, School of Medicine, Department of Critical Care and Pulmonary Services, Athens, Greece, 5Institut for Organische Chemie, Universität Leipzig, Leipzig, Germany, 6University of Athens, Medical School, Second University Department of Cardiology, Athens, Greece

Background: H2S is produced continuously at low levels in biological systems playing an important role in the regulation of cardiovascular function1. The diffusion ability of this gaseous molecule turns it into an attractive pharmacological agent for cardioprotection. Exogenous administration of rapid releasing H2S donors affords cardioprotection during ischemia/ reperfusion injury2.

Purpose: Pharmacological characterization of newly synthetized thioaminoacids (thioglycine, L-thiovaline and L-thiolysine) as slow releasing H2S donors. Further investigation of the cardioprotective role of thioglycine administrated in a rabbit model of ischemia/reperfusion injury and study of the underlying molecular mechanism(s) involved.

Methods: H2S release was determined through methylene blue method and a fluorescence- based assay3. Changes in cGMP levels in smooth muscle cells were measured by EIA in the absence of a phosphodiesterase inhibitor. In vivo infarct size was determined in 4 groups of anesthetized rabbits subjected to 30 minutes ischemia and 3 hours reperfusion: 1) Control group, no further intervention, 2) Thioglycine group, thioglycine was administrated at a dose of 16.26 μg*kg-1 bolus on the 20th min of ischemia followed by infusion of 0.16226 mg*kg-1 *h-1 for the next 120 min, 3) NaHS group, NaHS was administrated at a dose of 100 μg*kg-1 bolus on the 20th min of ischemia followed by infusion of 1 mg*kg-1 *h-1 for the next 120 min and 4) PostC group, animals were subjected to 8 cycles of 30sec ischemia/30sec reperfusion immediately after sustained ischemia. Drugs were administrated in saline. Dose of thioglycine was estimated as 1/10 equimolar of cardioprotective dose of NaHS4. The ratio of the infarct size (I) and the corresponding area at risk (R) was expressed as % I/R. Additional rabbits were subjected to the previous interventions up to 10th min of reperfusion for Akt, eNOS and GSK3β assessment.

Results: Thioglycine released more H2S than L-thiolysine and L-thiovaline reaching a plateau after 60 min, in contrast to the rapid rate observed with NaHS. Exposure of cultured rat aortic smooth muscle to thioaminoacids led to a concentration-dependent increase in cGMP levels. Exposure to L-thiolysine and thioglycine had a much more robust effect on cGMP levels than NaHS. Glycine, valine and lysine failed to increase cGMP levels. In the in vivo model of ischemia/reperfusion the following results were obtained:

Conclusion: Thioaminoacids liberate H2S at a slow rate versus inorganic salts and enhanced cGMP formation. Thioglycine triggered pharmacological postconditioning in rabbits during myocardial ischemia/reperfusion. The cytoprotective mechanism may involve activation of Akt and occurs independently of GSK3β and eNOS.

References: (1) Wang R, Physiol Rev 92: 791, 2012

(2) Lefer DJ et al, Clin Sci 120:219, 2011

(3) Zhou Z et al, Bioorg Med Chem 20:2675, 2012

(4) Bibli et al, ESC 2013, Amsterdam

C.C. was funded by a Postdoctoral Fellowship of the AHA (12POST12060643). This work has been co-financed by the EU (ESF) and Greek national funds through the Operational Program Education and Lifelong Learning of the (NSRF) - Research Funding Program: Thalis (MIS380259).